Chiral capillary electrophoretic determination of the enantiomeric purity of tetrahydronaphthalenic derivatives, melatoninergic ligands, using highly sulfated beta-cyclodextrins

Using cyclodextrin capillary zone electrophoresis (CD-CZE), baseline separation of synthetic tetrahydronaphthalenic derivatives, potential melatoninergic compounds, was achieved. A method for the enantioresolution of these tetralins and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters such as the nature and concentration of the chiral selectors, buffer pH, organic modifiers, temperature and applied voltage were investigated. The use of charged CDs provides a driving force for our neutral compounds in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-beta-CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution of three tetralin compounds was obtained using 25 mM phosphate buffer at pH 2.5 containing 2.5% w/v of highly S-beta-CD at 25 degrees C with an applied field of 0.25 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of linearity, sensitivity, accuracy and recovery. The enantiomeric purity for the three molecules was determined and the detection limit of enantiomer impurities is about 0.3-0.6%.     

Chiral capillary electrophoretic resolution of baclofen, gabaergic ligand, using highly sulfated cyclodextrins

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen, a potent GABA(B) agonist; was achieved. A method for the enantioresolution of this gamma-aminobutyric acid (GABA) and determination of enantiomeric purity was developed using CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters, such as the nature and concentration of the chiral selectors, buffer concentration, organic modifiers, and applied voltage, were investigated. The use of charged CDs provides a driving force in the opposite direction of the positively charged baclofen in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. Highly S-beta-CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution was obtained using 25 mM phosphate buffer, pH 2.5, containing 3% w/v highly S-beta-CD at 25 degrees C with aN applied field of 0.40 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of repeatability and limits of detection (0.13 microg x mL(-1)) and quantification. The migration order was determined.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.     

Enantioseparation of new nucleoside analogs, related to d4T and acyclovir, by chiral capillary electrophoresis using highly sulfated beta-cyclodextrins

Baseline separation of some new acyclic nucleosides which are potential antiviral agents was achieved using cyclodextrin capillary zone electrophoresis (CD-CZE). A method for the enantiomeric resolution of these compounds and determination of their enantiomeric purity was developed using anionic CDs (highly sulfated-CD or highly S-CD) as chiral selectors and capillaries, which were dynamically coated with polyethylene oxide (PEO). Operational parameters including (i) the nature and concentration of the chiral selectors, (ii) organic modifiers, (iii) temperature, and (iv) applied voltage were investigated. The use of charged CDs provides (i) a supplementary driving force for the compounds in a running buffer and (ii) enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S-CD was found to be the most effective complexing agent and allowed good enantiomeric resolution. The complete resolution of five nucleoside analogs was obtained using 25 mM phosphate buffer, pH 2.5, containing either highly S-alpha-CD, S-beta-CD or S-gamma-CD at 30 degrees C with an applied field of 0.30 kV/cm. The apparent association constants of the inclusion complexes were calculated. The enantiomer migration order for the molecules investigated was determined and the detection limit of enantiomeric impurities was found to vary between 0.34 to 3.56 ng.mL(-1) for the first enantiomer.     

Determination of brompheniramine enantiomers in rat plasma by cation‐selective exhaustive injection and sweeping cyclodextrin modified electrokinetic chromatography method

A method consisting of cation‐selective exhaustive injection and sweeping (CSEI‐sweeping) as online preconcentration followed by a cyclodextrin modified electrokinetic chromatography (CDEKC) enantioseparation has been developed for the simultaneous determination of two brompheniramine enantiomers in rat plasma. In this method, analytes were electrokinetically injected at a voltage of 8 kV for 80 s in a fused‐silica capillary. Prior to the injection, the capillary was rinsed with 50 mM phosphate buffer of pH 3.5, followed by a plug of a higher conductivity buffer (150 mM phosphate pH 3.5, 20 psi, 6 min) and a plug of water (0.5 psi, 5 s). Separation was carried out applying –20 kV in 50 mM phosphate buffer, pH 3.5, containing 10% v/v ACN and 30 mg/mL sulfated‐β‐cyclodextrin (S‐β‐CD). Analytical signals were monitored at 210 nm. The detection sensitivity of brompheniramine enantiomers was enhanced by about 2400‐fold compared to the normal injection mode (hydrodynamic injection for 3 s at 0.5 psi, with a BGE of 50 mM phosphate buffer containing 20 mg/mL S‐β‐CD at pH 3.5), and LLOQ of two enantiomers were both 0.0100 μg/mL. In addition, this method had fairly good repeatability and showed promising capabilities in the application of stereoselective pharmacokinetic investigations for brompheniramine enantiomers in rat.     

Optimization of sample stacking for the simultaneous determination of nonsteroidal anti-inflammatory drugs with a wall-coated histidine capillary column

A wall-coated histidine capillary column was developed for the on-line preconcentration of nonsteroidal anti-inflammatory drugs (NSAIDs) in capillary electrochromatography (CEC). A wide variety of experimental parameters, such as the sample buffer, background electrolyte (BGE) composition, concentration, sample plug lengths, water plug, and the effect of organic modifiers were studied. The relationship between peak height and injection times for the NSAIDs by variation of sample and BGE buffer concentration was investigated. On addition of sodium chloride (0.3-0.6%) to the sample zone, the stacking efficiency was increased. With acetate buffer (100 mM, pH 5.0)/ethanol (20% v/v) as BGE and sample solution in acetate buffer (0.2 mM, pH 5.0)/ethanol (20% v/v)/NaCl (0.3% w/v), NSAIDs could be determined at low microM levels without sample matrix removal. The detection limit was 0.096 microM for indoprofen, 0.110 microM for ketoprofen, 0.012 microM for naproxen, 0.023 microM for ibuprofen, 0.110 microM for fenoprofen, 0.140 microM for flurbiprofen, and 0.120 microM for suprofen. The method could be successfully applied to the simultaneous determination of NSAIDs in urine. The recoveries were better than 82% for all the analytes. The present method enables simple manipulation with UV detection for the determination of NSAIDs at low concentration levels in complex matrix samples.     

Chiral resolution of melatoninergic ligands by EKC using highly sulfated CDs

EKC methods for the enantiomeric resolutions of melatoninergic ligands were developed using anionic CDs (highly S-alpha-CD, highly S-beta-CD, and highly S-gamma-CD) as chiral selectors at acidic pH 2.5. The optimization of the various operational parameters (nature and concentration of the CD, phosphate buffer concentration, addition of organic modifiers in the BGE, and temperature) allows baseline enantioresolutions (superior to 2) in short analysis times (inferior to 7 min) for all studied analytes. Some analytical characteristics of the optimal method were then studied for each analyte: repeatability, linearity, and LOD and LOQ. Lastly, determination of the apparent binding constants for the 18 complexes formed between the six analytes and the three CDs led us to rationalize the complexation mechanisms.     

Cation-selective exhaustive injection and sweeping MEKC for direct analysis of methamphetamine and its metabolites in urine

Direct analysis of methamphetamine, amphetamine, and p-hydroxymethamphetamine in urine was achieved by cation-selective exhaustive injection and sweeping micellar EKC. A bare fused-silica capillary (40 cm, 50 microm id) was filled with phosphate buffer (80 mM, pH 3, containing 20% ACN). Then a high-conductivity buffer (100 mM phosphate, pH 3; 6.9 kPa for 2.5 min) was injected. Samples were loaded using electrokinetic injection (10 kV, 600 s) which created long zones of cationic analytes. To enhance sensitivity by sweeping, the stacking step was performed using a phosphate buffer (50 mM, pH 3, containing 20% ACN and 100 mM SDS) at -20 kV before separation by MEKC. This method was capable of detecting the analytes at ppb levels. The calibration plots were linear (r(2) >or= 0.9948) over a range of 100-5000 ng/mL for methamphetamine, and 100-2000 ng/mL for amphetamine and p-hydroxymethamphetamine. The LODs (S/N = 3) were 20 ng/mL for methamphetamine, and 15 ng/mL for amphetamine and p-hydroxymethamphetamine. The method was applied to analysis of 14 urine samples of addicts and is suitable for screening suspected samples for forensic purposes. The results showed good agreement with fluorescence polarization immunoassay and GC-MS.     

Determination of anthracycline antibiotics doxorubicin and daunorubicin by capillary electrophoresis with UV absorption detection.

Sweeping preconcentration and electrokinetic injection was used for the capillary electrophoretic analysis of trace amounts of biologically active anthracyclines with UV absorption detection. Phosphate buffer (100 mM), pH 2.5, with addition of 40% v/v methanol was used as background electrolyte (BGE). Sodium dodecyl sulfate (150 mM) was added to BGE in the inlet vial as the sweeping agent. The system enables effective separation of anthracyclines as well as cleanup from matrix impurities. Sweeping preconcentration of sample provides an excellent detection limit (1 x 10(-9) mol L(-1)). The method was applied for the determination of therapeutic levels of doxorubicin in real plasma samples.     

Analysis of metformin dosage formulations by capillary electrophoresis at nano scale detection

An inexpensive, rapid and reproducible capillary electrophoretic method has been developed and validated for the determination of metformin in pharmaceutical preparations. The method was developed utilizing a fused silica capillary (60 cm x 50 microm I.D.), phosphate buffer (50 mM, 3.0 pH)-acetonitrile (95:5, v/v) as background electrolyte (BGE), 20 kV applied voltage with UV detection at 254 nm and at a working temperature of 23 +/- 1 degrees C. Linearity was observed in the concentration range from 100 ng/L to 5 microg/L, with a correlation coefficient (R2) of 0.9998. The limits of detection and quantification achieved were 60 and 100 ng/mL, respectively. The recovery of metformin from pharmaceutical preparations was 99.1%. These validation parameters demonstrate the precision of the method and its suitability for the determination of metformin in pharmaceutical tablet formulations.     

Development of an ultrasensitive stacking technique for 5‐nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R² ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.     

Development and validation of capillary electrophoresis method for tobramycin with precapillary derivatization and UV detection

One of the major drawbacks in the analysis of aminoglycoside antibiotics is their lack of UV chromophore and/or fluorophore. Tobramycin, a representative member of this group, was examined in this study. To overcome the detection hurdle, a precapillary derivatization followed by capillary electrophoresis analysis with direct UV detection was investigated. A central composite design was applied to optimize the method and three parameters were selected in this study: buffer pH, temperature and % acetonitrile (ACN). Selectivity between tobramycin main component and its adjacent peaks as well as the peak efficiency and symmetry factors were established as responses. For each response, a model was obtained by a second-order mathematical expression. Successful results were obtained with a simple background electrolyte (BGE) containing 30 mM sodium tetraborate, pH 10.2, and ACN (75:25 v/v). Under these conditions, baseline separation of tobramycin from its adjacent kanamycin B and an unknown peak was achieved. A temperature of 20 degrees C and applied voltage of 28.0 kV were used. The method showed good validation data in terms of precision, limits of quantitation and detection, specificity and linearity and was found to be suitable for analysis of tobramycin bulk pharmaceutical samples.     

Enantiomeric analysis of baclofen analogs by capillary zone electrophoresis, using highly sulfated cyclodextrins: Inclusion ionization constant pKa determination

Using cyclodextrin-capillary zone electrophoresis (CD-CZE), baseline separation of baclofen phaclofen, saclofen, and hydroxy-saclofen, potent gamma-aminobutyric acid(B) (GABA(B)) agonist or antagonists was achieved. A method for the enantioresolution of those analogs of GABA was developed using anionic cyclodextrins (highly sulfated CD or highly S-CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). With charged CDs we observed good resolutions due to the large electrophoretic mobility of these chiral selectors opposite to the mobility of the solutes. The highly S-alpha-CD and S-beta-CD were found to be complementary and the most effective complexing agent, allowing good enantiomeric resolution in short runtimes. The complete resolution was obtained using 25 mM phosphate buffer at pH 2.5 containing 3% w/v of highly S-alpha-CD or S-beta-CD at 25 degrees C with an applied field of 0.30 kV/cm. The apparent binding constants of the inclusion complexes were evaluated and the migration order was determined. A comparison was possible to investigate the importance of the anionic group of the molecules in the separations. The pK(a) values were determined for all four compounds in order to explain relative electrophoretic migration of the solutes.     

Simultaneous determination of seven flavonoids in Epimedium using pressurized liquid extraction and capillary electrochromatography

Herba Epimedii (known as Yinyanghuo in China) is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a CEC method was developed for the simultaneous determination of seven flavonoids, including hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, icariin, epimedin A, B, and C, in Epimedium using baicalein as internal standard (IS). The influence of relevant parameters such as buffer concentration, pH, and proportion of ACN was investigated and optimized. Baseline separation was obtained using a Hypersil C18 capillary (3 microm, 100 microm/25 cm) with a mixture of 20 mM phosphate buffer (pH 4.0)/ACN (70:30 v/v) as mobile phase running at 30 kV and 25 degrees C in 20 min. All calibration curves showed good linearity (r2 >0.9992) within test ranges. The LOD and LOQ were lower than 8.6 and 42.8 microg/mL, respectively. The RSDs of intra- and interday for relative peak areas of seven analytes were less than 3.1 and 4.4%, and the recoveries were 95.2-103.3%. Samples of different Epimedium species were analyzed using the validated method, which is useful for quality control of Epimedium and its medical preparations.     

Determination of the enantiomeric and diastereomeric impurities of RS‐glycopyrrolate by capillary electrophoresis using sulfated‐β‐cyclodextrin as chiral selectors

A practical chiral CE method, using sulfated‐β‐CD as chiral selector, was developed for the enantioseparation of glycopyrrolate containing two chiral centers. Several parameters affecting the separation were studied, including the nature and concentration of the chiral selectors, BGE pH, buffer type and concentration, separation voltage, and temperature. The separation was carried out in an uncoated fused‐silica capillary of (effective length 40 cm) × 50 μm id with a separation voltage of 20 kV using 30 mM sodium phosphate buffer (pH 7.0, adjusted with 1 M sodium hydroxide) containing 2.0% w/v sulfated‐β‐CD at 25°C. Finally, the method for determining the enantiomeric impurities of RS‐glycopyrrolate was proposed. The method was further validated with respect to its specificity, linearity range, accuracy and precision, LODs, and quantification in the expected range of occurrence for the isomeric impurities (0.1%).     

Chiral separation of cetirizine by capillary electrophoresis

Chiral separation of cetirizine, a second-generation H(1)-antagonist, was studied by CD-mediated CE. Several parameters, including pH, CD type, buffer concentration, type of co-ion, applied voltage and temperature, were investigated. The best conditions for chiral separation were obtained using a 75 mM triethanolamine-phosphate buffer (pH 2.5) containing 0.4 mg/mL heptakis(2,3-diacetyl-6-sulfato)-beta-CD and 10% ACN. Online UV detection was performed at 214 nm, a voltage of 20 kV was applied and the capillary was temperature controlled at 25 degrees C by liquid cooling. Hydrodynamic injection was performed for 1 s. The method was validated for the quantification of levocetirizine in tablets and for enantiomeric purity testing of the drug substance. Selectivity, linearity, LOD and LOQ, precision and accuracy were evaluated for both methods. The amount of levocetirizine dihydrochloride in the commercially available tablets was quantified and was found to be within the specification limits of the claimed amount (5 mg). The amount of distomer in levocetirizine drug substance was found to be 0.87 +/- 0.09% w/w, which is in agreement with the certificate of analysis supplied by the company.     

Analysis of red inks by micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection

A combination of MEKC with a—highly sensitive but not commonly used—LIF detector was tested regarding the possibility of differentiation of red inks. The separation process was conducted in a fused silica capillary (id 50 μm, 60/50 cm total/effective length) in BGE consisting of 40 mM sodium borate, 20 mM SDS and 10% v/v ACN with 30 kV applied. The optimized temperature of storage and capillary was 10 and 25°C, respectively. The samples were prepared using 20 dots (ø 0.5 mm), extracted in 35 μL BGE and diluted with 30 μL of water. The proposed method showed excellent repeatability and reproducibility (RSD (t_m) < 0.8 and < 2.5%, respectively). It was applied to group identification and differentiation of different brands, models, and batches of red printing, stamp, and pen inks. It was demonstrated that differentiation can be performed effectively on the basis of migration times and ratios of peak areas. The high efficiency of the developed method was indicated by discriminating power ranging from 87.3 to 98.8%, for stamp and pen inks, respectively. The results showed that the proposed procedure can be valuable for an objective examination of the red parts of questioned documents.     

Quantification of buserelin in a pharmaceutical product by multiple-injection CZE

An efficient and rapid separation method based on reversed-polarity multiple-injection CZE (MICZE), has been developed for the quantification of buserelin in a pharmaceutical product. The determinations were performed by serially injecting five standard solutions of buserelin (50-300 microg/mL) and one reference analyte into a Polybrene-coated capillary. All the samples contained goserelin, an analog peptide to buserelin, as internal standard (IS). Immediately after pressure injection, the applied sample plugs were subjected to electrophoresis for 2 min at -25 kV. Consequently, each sample plug became isolated from its neighboring plugs by the BGE, composed of 100 mM phosphate-triethanolamine buffer at pH 3.0 containing 10% v/v ACN. During separation the individual sample components migrated at similar velocities and as distinct zones through the capillary giving 24 peaks, 12 from the analyte and the IS and 12 from the sample matrix. The buserelin content of the pharmaceutical product was determined to be 0.94 +/- 0.05 mg/mL, which is only a slight deviation from the declared concentration (1 mg/mL).     

Enantioseparation of chiral N-imidazole derivatives by electrokinetic chromatography using highly sulfated cyclodextrins: mechanism of enantioselective recognition

Baseline separation of ten new substituted [1-(imidazo-1-yl)-1-phenylmethyl)] benzothiazolinone and benzoxazolinone derivatives, with one chiral center, was achieved by CD-EKC using highly sulfated CDs (alpha, beta, gamma highly S-CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The highly S-CDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times inferior to 2.5 min and resolution factors R(s) of 3.73, 3.90, 1.40, and 4.35 for compounds 1, 2, 3, and 5, respectively, using 25 mM phosphate buffer at pH 2.5 containing either highly S-alpha-CD, highly S-beta-CD, and highly S-gamma-CD (3 or 4% w/v) at 298 K, with an applied field of 0.30 kV/cm. The determination of the enantiomer migration order for the various analytes and the study of the analyte structure-enantioseparation relationships display the high contribution of the interactions between the analytes phenyl ring and the CDs to the enantiorecognition process. The thermodynamic study of the analyte-CD affinities permits us to improve our knowledge about the enantioseparation mechanism.     

Development and validation of a capillary electrophoresis method for the determination of escitalopram and sensitive quantification of its enantiomeric impurity in formulations

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R2 = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).